Electron Microscopy - Union Collegeminerva.union.edu/newmanj/Physics200/EM.pdf · Electron...
Transcript of Electron Microscopy - Union Collegeminerva.union.edu/newmanj/Physics200/EM.pdf · Electron...
Electron Microscopy Chelsea Aitken Peter Aspinall
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Intro to Electron Microscopy
Similar to optical microscopy except with electrons rather than photons
Used to image samples with a resolution of 10 Å Can image many different structural geometries
Mostly limited by radiation damage from the electron beam
Electron Properties Since electrons exhibit wave and particle
behavior, the de Broglie relationship applies: ℎ = λ𝑒p
Since the electron is charged, when introduced to an electric potential difference, it accelerates to its equilibrium momentum:
𝑞φ =12𝑝2
𝑚 → 𝑝 = 2𝑚𝑒𝑒φ
So particle momentum is only dependent on the electric potential difference
Electromagnetic Lenses Used to focus the electron beam We can relate wavelength to accelerating voltage:
λ𝑒 =ℎ
2𝑚𝑒𝑒φ≈ 12φ−
12
Electron wavelengths are 5 times smaller photons Maximum resolution (d) of a lens is related to the
aperture angle and the wavelength by:
𝑑 =λ𝑒
sin𝛼
However due to aberration in the lens, the resolution is also limited by:
𝑑 = 𝐶𝑠𝜆𝑒3 1 4⁄ Where Cs is the spherical aberration coefficient
Signal vs. Noise Largest issue is radiation damage to the specimen Image is generated from elastic scattering while noise is
generated from the inelastic scattering Inelastic scattering deposits energy on the sample which
damages the sample (occurs 3-4 times more often than elastic scattering events)
Signal-to-noise ratio is described by the Rose model:
𝑆 𝜎𝑅𝑅𝑠𝑒⁄ = 𝐶 𝐴𝑛𝑏𝑏𝑏𝑏𝑏𝑏𝑅𝑏𝑏𝑏
Where C is the contrast (C = 𝑏𝑜𝑜𝑜𝑜𝑜𝑜−𝑏𝑜𝑏𝑜𝑏𝑏𝑏𝑜𝑏𝑏𝑏𝑏𝑜𝑏𝑜𝑏𝑏𝑏𝑜𝑏𝑏𝑏� )
Ratios between 5-7 are required to identify features with good enough confidence
Can either use an energy filter (remove certain energies) or higher accelerating voltage
Effect of the Microscope on Electron When a wave is passed through a samples, it interacts
and is released as a phase shift of the wave in. This is represented by:
𝜏𝑅𝑏𝑜 𝑥,𝑦 = 𝜏𝑖𝑏𝑒𝑖φ𝑃(𝑥,𝑦) ≈ 𝜏𝑖𝑏(1 + 𝑖φ𝑃 𝑥, 𝑦 ) This is then adjusted to account for electron
absorbance: 𝜏𝑅𝑏𝑜 𝑥, 𝑦 = 𝜏𝑖𝑏(1 + 𝑖φ𝑃 𝑥,𝑦 + 𝜇(𝑥, 𝑦))
The microscope then observes the phase shift due to this change which can be represented by the Fourier Transform: 𝑇𝑅𝑏𝑜(𝑢,𝑣) = 𝜏𝑖𝑏[𝛿 𝑢, 𝑣 + 𝑖Φ𝑃 𝑢, 𝑣 + Μ 𝑢, 𝑣 ] × 𝑃(𝑢,𝑣)
Where P(u,v) is the transfer function of the microscope
Electron Generation
Thermionic Electron Gun Heated filament produces
electrons Typically made of Tungsten or
Lanthanum hexaboride Electrons drawn towards an
anode An aperture in the anode
creates a beam
Field Emission Gun A very strong electric field is
used to extract electrons from a metal filament Filament typically a single
tungsten crystal Requires a vacuum Similar anode setup
Microscope Setup Transmission Electron
Microscope Phase contrast Image is
formed by the interference between electrons that passed through the sample and ones that did not
Scanning Electron Microscope Electron beam is scanned
across the sample The reemitted electrons are
measured in order to form the image
Focusing
When the image is in focus, there is very low contrast due to the electron loss around the objective
By imaging underfocus or overfocus, a phase shift and amplitude contrast are created
This creates a dark image with a white ring around or a white image with a dark ring (respectively
Negative Staining Biological samples are
often imaged using negative staining
The elements of biological molecules do not interact strongly with the electron beam
Instead they are seated in a material that does and then the negative space of the sample is imaged in this material
http://www.izw-berlin.de/electron-microscopy.html
Cryo-Microscopy Samples are often frozen in
order to preserve the structure against radiation damage from the electron beam
In order to not damage the structure when freezing, the sample is flash frozen If ice crystals were allowed to
form they would damage the sample
Samples are typically dunked into liquid ethane or propane (~11o K)
Image Options EM can magnify a sample between 1000 and 200,000
times However due to limitations, macromolecules are usually
imaged between 40,000 and 60,000 for a resolution of 10 – 20 Å
Image intensity decreases as magnification goes up with 1/M2
Protein concentration is typically around 1 mg ml-1 to ensure sufficient particle density without being overcrowded
Biological samples can only be exposed to 10-15 electrons per Å2 Using a stain allows increased exposure Lower temperatures can similarly protect the sample
Data Collection Protocol Search/Focus/Exposure
Use low-dose/magnification to find area of interest to magnify
Specific defocus is picked and drift is checked
Sample is exposed to a high-does to image the sample
Imaging Symmetry When molecules have
symmetry or are in helical structures, a two dimensional EM image can be used to reconstruct the 3D structure
This information is often using in conjunction with X-ray crystallography in determined the crystal structure of molecules
http://www.newscientist.com/data/images/ns/cms/dn22545/dn22545-1_300.jpg
Electron Tomography Data is collected
at multiple tilt angles Typically every 1o –
2o over + 70o
Image is then compiled to determine the 3D structure of the image
http://origin-ars.els-cdn.com/content/image/1-s2.0-S0301462202003071-gr1.jpg
Immunochemical Applications It is very easy to image
gold clusters with EM due to gold’s properties
Thus the use of gold labeled antibodies is particularly helpful in immunochemistry
Labeled antibodies will bind to their antigen
EM can then be used to identify the location of antibodies and by extension the antigens
http://www.nano.org.uk/news/images/imageL1282120449.jpg
Sources 1. Serdyuk, Igor N., Nathan R. Zaccai, and
Joseph Zaccai. Methods in Molecular Biophysics: Structure, Dynamics, Function. New York: Cambridge University Press, 2007. Print.
2. "Introduction to Electron Microscopes." Matter.org.uk. University of Liverpool, n.d. Web. 20 Oct 2013. <http://www.materials.ac.uk/elearning/matter/IntroductionToElectronMicroscopes/SEM/electron-gun.html>.